RESUMEN

In mammals, activation of primordial follicles to primary follicle is a progressive and highly regulated process. There is evidence in mice that phosphatase and tensin homologue deleted on Chromosome 10 (PTEN) silencing is an important negative regulator of phosphatidylinositol 3-kinase (PI3K), which initiates activation of dormant follicles. The objective of the study was to evaluate the effect of the addition of PTEN inhibitor (bpV(HOpic)) (10 µM) and/or Kit Ligand (KL) (100 ng/mL) on the in vitro activation and survival of alpaca primordial follicles. Ovarian cortical fragments from 11 adult alpacas were cultured for 24 h in tissue culture medium (α-MEM+ ) supplemented with KL and bpV or the association of both. Subsequently, each sample was processed by classical histology and follicular counting and classification were performed. The results obtained show a reduction (p < 0.05) of primordial follicles in more than 50% in follicular tissue cultured in vitro in α-MEM+ or supplemented with bpV and/or KL versus the control (not cultured). Further, >25% increase in primary follicles in follicular tissue cultured in vitro in α-MEM+ or supplemented with KL and/or bpV versus control. However, the follicular survival rate showed a decrease of 20% in the cultured tissues, except for the α-MEM+ supplemented with KL and bpV. In conclusion, supplementation of bpV (HOpic) (10 µM) and KL (100 ng/mL) increased the activation in vitro of primordial follicles and survival after in vitro culture of alpaca ovarian tissue.

Asunto(s)

Camélidos del Nuevo Mundo , Femenino , Animales , Ratones , Factor de Células Madre/farmacología , Fosfatidilinositol 3-Quinasas/farmacología , Folículo Ovárico/fisiología

RESUMEN

As an important component of solid tumors, mast cells show specific phenotypes in various tumor microenvironments. However, the precise mechanism of mast cell accumulation and the phenotypic features of thyroid cancer (TC) remain largely unknown. Here, we found that mast cells were obviously recruited to tumor tissue by TC-derived stem cell factor (SCF). With tumor progression, mast cell levels increased gradually. In addition, intratumoral mast cells expressed higher levels of the immunosuppressive molecule galectin-9, which effectively suppresses CD8+ T-cell antitumor immunity in vitro. Blocking galectin-9 on tumor-infiltrating mast cells reversed the immunosuppression of CD8+ T cells. In conclusion, our data elucidated novel protumorigenic and immunosuppressive roles of mast cells in TC. In addition, our results indicated that blocking mast cells may impede tumor progression and ameliorate the prognosis of TC patients.

Asunto(s)

Mastocitos , Neoplasias de la Tiroides , Humanos , Mastocitos/patología , Linfocitos T CD8-positivos , Factor de Células Madre , Galectinas , Microambiente Tumoral

RESUMEN

Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07 ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses. KEY POINTS: • Soluble glycosylated human SCF exerted improved proliferative activity on UT-7 cells. • Three mAbs with high specificity targeting glycosylated human SCF were obtained. • mAbs applications comprise sandwich ELISA, western blot, and immunofluorescence assays.

Asunto(s)

Anticuerpos Monoclonales , Glicoproteínas , Hibridomas , Factor de Células Madre , Humanos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Biotecnología , Glicoproteínas/inmunología , Células HEK293 , Factor de Células Madre/análisis , Factor de Células Madre/inmunología , Glicosilación , Ensayo de Inmunoadsorción Enzimática , Western Blotting

RESUMEN

Background Melasma is an acquired dyschromia with several histologic alterations in the epidermis, basement membrane and upper dermis. The treatment of melasma is challenging due to the irregular response and chronicity of the disease. To date, there are no curative strategies, largely due to the limited understanding of the intrinsic effects of each treatment. Objectives The objective of the study was to evaluate the histological changes promoted by triple combination cream, with or without complementary treatment with microneedling and oral tranexamic acid, in the treatment of melasma. Methods A factorial, randomised, controlled and evaluator-blinded clinical trial was performed involving 64 women with facial melasma, divided in four groups, who underwent 60 days of treatment with triple combination cream alone (control group) or combined with two monthly microneedling sessions (microneedling group), TA 250 mg twice daily (tranexamic acid group), or both tranexamic acid group and microneedling group. The participants underwent biopsy of the area with melasma at inclusion (D1) and D60. The primary outcomes were the variation (D1 × D60) between the variables: Thickness of the epidermis and stratum corneum, stratum corneum compaction and solar elastosis; melanin density in the epidermis and upper dermis; proportion between the extension of the nonintact and intact basement membrane zone; mast cell count in the upper dermis; melanocyte count in the basal layer, pendulum melanocyte count and melanocyte area; immunostaining density of vascular endothelial growth factor; stem cell factor and keratinocyte growth factor. Results One participant in the TG discontinued tranexamic acid due persistent headache; and herpes simplex occurred in three patients after microneedling. The groups showed a 24% (CI95%: 17-35%; P < 0.01) reduction in epidermal melanin density. There was no change in dermal melanin density or the area of melanocytes after treatment. There was an overall 25% (CI95%: 7-42%; P < 0.01) reduction in the number of pendulum melanocytes, especially in the microneedling and tranexamic acid group, that presented a 41% (CI95%: 7-73%; P < 0.01) reduction. The extension of the nonintact basal membrane relative to the intact basal membrane decreased after treatment, especially in microneedling group and microneedling and tranexamic acid group. There was an increase of 13% (CI95%: 5-21%; P = 0.02) in epidermal thickness and 6% (CI95%: 0-22%; P = 0.04) thinning of the stratum corneum in the groups. All groups showed stratum corneum compaction. Solar elastosis improved only in the microneedling group and microneedling and tranexamic acid group. Vascular endothelial growth factor immunostaining increased 14% (CI95%: 4-24%; P = 0.03) in the groups; and stem cell factor increased only in microneedling group. There was no change in the number of mast cells, CD34 and keratinocyte growth factor immunostaining. Limitations The site of biopsy may not represent all of the facial melasma and the immunohistochemical sensitivity of the cytokines does not have a stoichiometric relationship with proteins. Conclusion A greater thickness of the epidermis is associated with melasma bleaching. Dermal melanin seems to have no impact on melasma prognosis. Damage to the skin barrier and stimulus of angiogenesis should be avoided in the treatment of melasma. Microneedling complements the topical treatment of melasma by improving patterns of skin photoaging. Oral tranexamic acid complements the topical treatment of melasma by inhibiting the stem cell factor.

Asunto(s)

Melanosis , Ácido Tranexámico , Humanos , Femenino , Factor 7 de Crecimiento de Fibroblastos/uso terapéutico , Melaninas , Factor A de Crecimiento Endotelial Vascular , Factor de Células Madre/uso terapéutico , Melanosis/terapia , Melanosis/tratamiento farmacológico , Resultado del Tratamiento

RESUMEN

Innate lymphoid cells (ILCs) are the most recently described group of lymphoid subpopulations. These tissue-resident cells display a heterogeneity resembling that observed on different groups of T cells, hence their categorization as cytotoxic NK cells and helper ILCs type 1, 2 and 3. Each one of these groups is highly diverse and expresses different markers in a context-dependent manner. Type 2 innate lymphoid cells (ILC2s) are activated in response to helminth parasites and regulate the immune response. They are involved in the etiology of diseases associated with allergic responses as well as in the maintenance of tissue homeostasis. Markers associated with their identification differ depending on the tissue and model used, making the study and understanding of these cells a cumbersome task. This review compiles evidence for the heterogeneity of ILC2s as well as discussion and analyses of molecular markers associated with their identity, function, tissue-dependent expression, and how these markers contribute to the interaction of ILC2s with specific microenvironments to maintain homeostasis or respond to pathogenic challenges.

Asunto(s)

Antígenos de Diferenciación/análisis , Subgrupos Linfocitarios/inmunología , Tejido Adiposo Blanco/inmunología , Tejido Adiposo Blanco/patología , Animales , Citocinas/metabolismo , Helmintiasis/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Homeostasis , Humanos , Inmunofenotipificación , Inflamación , Intestinos/inmunología , Pulmón/inmunología , Subgrupos Linfocitarios/química , Ratones , Nutrientes , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-kit/inmunología , Receptores de Superficie Celular/inmunología , Piel/inmunología , Factor de Células Madre/inmunología

RESUMEN

Supplements containing pharmacological concentrations of biotin are commercially available. The mechanisms by which biotin at pharmacological concentrations exerts its action have been the subject of multiple investigations, particularly for biotin's medicinal potential and wide use for cosmetic purposes. Several studies have reported that biotin supplementation increases cell proliferation; however, the mechanisms involved in this effect have not yet been characterized. In a previous study, we found that a biotin-supplemented diet increased spermatogonia proliferation. The present study was focused on investigating the molecular mechanisms involved in biotin-induced testis cell proliferation. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for eight weeks. Compared with the control group, the biotin-supplemented mice presented augmented protein abundance of the c-kit-receptor and pERK1/2Tyr204 and pAKTSer473, the active forms of ERK/AKT proliferation signaling pathways. No changes were observed in the testis expression of the stem cell factor and in the serum levels of the follicle-stimulating hormone. Analysis of mRNA abundance found an increase in cyclins Ccnd3, Ccne1, Ccna2; Kinases Cdk4, Cdk2; and E2F; and Sp1 & Sp3 transcription factors. Decreased expression of cyclin-dependent kinase inhibitor 1a (p21) was observed but not of Cdkn2a inhibitor (p16). The results of the present study identifies, for the first time, the mechanisms associated with biotin supplementation-induced cell proliferation, which raises concerns about the effects of biotin on male reproductive health because of its capacity to cause hyperplasia, especially because this vitamin is available in large amounts without regulation.

Asunto(s)

Biotina/toxicidad , Proliferación Celular/efectos de los fármacos , Suplementos Dietéticos/toxicidad , Hormona Folículo Estimulante/sangre , Espermatogonias/efectos de los fármacos , Factor de Células Madre/metabolismo , Testículo/efectos de los fármacos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Masculino , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/genética , Factor de Transcripción Sp3/metabolismo , Espermatogonias/metabolismo , Espermatogonias/patología , Testículo/metabolismo , Testículo/patología

RESUMEN

Degeneration of motor neurons, glial cell reactivity, and vascular alterations in the CNS are important neuropathological features of amyotrophic lateral sclerosis (ALS). Immune cells trafficking from the blood also infiltrate the affected CNS parenchyma and contribute to neuroinflammation. Mast cells (MCs) are hematopoietic-derived immune cells whose precursors differentiate upon migration into tissues. Upon activation, MCs undergo degranulation with the ability to increase vascular permeability, orchestrate neuroinflammation and modulate the neuroimmune response. However, the prevalence, pathological significance, and pharmacology of MCs in the CNS of ALS patients remain largely unknown. In autopsy ALS spinal cords, we identified for the first time that MCs express c-Kit together with chymase, tryptase, and Cox-2 and display granular or degranulating morphology, as compared with scarce MCs in control cords. In ALS, MCs were mainly found in the niche between spinal motor neuron somas and nearby microvascular elements, and they displayed remarkable pathological abnormalities. Similarly, MCs accumulated in the motor neuron-vascular niche of ALS murine models, in the vicinity of astrocytes and motor neurons expressing the c-Kit ligand stem cell factor (SCF), suggesting an SCF/c-Kit-dependent mechanism of MC differentiation from precursors. Mechanistically, we provide evidence that fully differentiated MCs in cell cultures can be generated from the murine ALS spinal cord tissue, further supporting the presence of c-Kit+ MC precursors. Moreover, intravenous administration of bone marrow-derived c-Kit+ MC precursors infiltrated the spinal cord in ALS mice but not in controls, consistent with aberrant trafficking through a defective microvasculature. Pharmacological inhibition of c-Kit with masitinib in ALS mice reduced the MC number and the influx of MC precursors from the periphery. Our results suggest a previously unknown pathogenic mechanism triggered by MCs in the ALS motor neuron-vascular niche that might be targeted pharmacologically.

Asunto(s)

Esclerosis Amiotrófica Lateral/inmunología , Mastocitos/inmunología , Microvasos/patología , Neuronas Motoras/patología , Enfermedades Neuroinflamatorias/inmunología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Médula Espinal/inmunología , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Astrocitos/metabolismo , Astrocitos/patología , Benzamidas/farmacología , Estudios de Casos y Controles , Quimasas/metabolismo , Ciclooxigenasa 2/metabolismo , Femenino , Humanos , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Microvasos/metabolismo , Persona de Mediana Edad , Neuronas Motoras/metabolismo , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Piridinas/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Factor de Células Madre/metabolismo , Tiazoles/farmacología , Triptasas/metabolismo

RESUMEN

Lymphoma is the most common type of canine hematological malignancy where the multicentric (cMCL) form accounts for 75% of all cases. The standard treatment is the CHOP chemotherapy protocols that include cyclophosphamide, doxorubicin, vincristine and prednisone, where the majority of dogs achieve complete/partial response; however, it is very important to predict non-responsive cases to improve treatment and to develop new targeted therapies. Here we evaluate a liquid biopsy approach based on serum Small Extracellular Vesicles enriched for exosomes (SEVs) to predict cMCL chemotherapy response. Nineteen dogs at the end of the 19-week chemotherapy protocol (8 Complete Response and 11 Progressive Disease) were evaluated for serum SEVs size, concentration and screened for 95 oncomirs. PD patients had higher SEVs concentration at the diagnosis than CR patients (P = 0.034). The ROC curve was significant for SEVs concentration to predict the response to CHOP (AUC = 0.8011, P = 0.0287). A potential molecular signature based on oncomirs from SEVs (caf-miR-205, caf-miR-222, caf-mir-20a and caf-miR-93) is proposed. To the best of our knowledge, this is the first study demonstrating the potential of a liquid biopsy based on SEVs and their miRNAs content to predict the outcome of chemotherapy for canine multicentric lymphomas.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biomarcadores de Tumor/genética , Enfermedades de los Perros/tratamiento farmacológico , Vesículas Extracelulares/genética , Linfoma/tratamiento farmacológico , Linfoma/veterinaria , MicroARNs/genética , Animales , Biomarcadores de Tumor/sangre , Estudios de Casos y Controles , Ciclofosfamida/farmacología , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/genética , Enfermedades de los Perros/mortalidad , Perros , Doxorrubicina/farmacología , Vesículas Extracelulares/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Biopsia Líquida , Linfoma/genética , Linfoma/mortalidad , Masculino , MicroARNs/sangre , Fosfatidilinositol 3-Quinasas/sangre , Fosfatidilinositol 3-Quinasas/genética , Prednisona/farmacología , Isoformas de Proteínas/sangre , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas c-kit/sangre , Proteínas Proto-Oncogénicas c-kit/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/sangre , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Recurrencia , Factor de Células Madre/sangre , Factor de Células Madre/genética , Análisis de Supervivencia , Resultado del Tratamiento , Vincristina/farmacología

RESUMEN

Distal axonopathy is a recognized pathological feature of amyotrophic lateral sclerosis (ALS). In the peripheral nerves of ALS patients, motor axon loss elicits a Wallerian-like degeneration characterized by denervated Schwann cells (SCs) together with immune cell infiltration. However, the pathogenic significance of denervated SCs accumulating following impaired axonal growth in ALS remains unclear. Here, we analyze SC phenotypes in sciatic nerves of ALS patients and paralytic SOD1G93A rats, and identify remarkably similar and specific reactive SC phenotypes based on the pattern of S100ß, GFAP, isolectin and/or p75NTR immunoreactivity. Different subsets of reactive SCs expressed colony-stimulating factor-1 (CSF1) and Interleukin-34 (IL-34) and closely interacted with numerous endoneurial CSF-1R-expressing monocyte/macrophages, suggesting a paracrine mechanism of myeloid cell expansion and activation. SCs bearing phagocytic phenotypes as well as endoneurial macrophages expressed stem cell factor (SCF), a trophic factor that attracts and activates mast cells through the c-Kit receptor. Notably, a subpopulation of Ki67+ SCs expressed c-Kit in the sciatic nerves of SOD1G93A rats, suggesting a signaling pathway that fuels SC proliferation in ALS. c-Kit+ mast cells were also abundant in the sciatic nerve from ALS donors but not in controls. Pharmacological inhibition of CSF-1R and c-Kit with masitinib in SOD1G93A rats potently reduced SC reactivity and immune cell infiltration in the sciatic nerve and ventral roots, suggesting a mechanism by which the drug ameliorates peripheral nerve pathology. These findings provide strong evidence for a previously unknown inflammatory mechanism triggered by SCs in ALS peripheral nerves that has broad application in developing novel therapies.

Asunto(s)

Esclerosis Amiotrófica Lateral/patología , Inflamación/metabolismo , Interleucinas/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Células de Schwann/metabolismo , Factor de Células Madre/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Axones/patología , Modelos Animales de Enfermedad , Humanos , Masculino , Neuronas Motoras/patología , Neuroglía/metabolismo , Ratas Transgénicas

RESUMEN

The objective was to study the effect of mechanical intestinal obstruction in rats on the phenotype of interstitial cells of Cajal (ICC). Healthy Wistar rats were randomly divided into sham-operation group (C), one day obstruction group (M1), two days obstruction group (M2), and three days obstruction group (M3), with 10 rats in each group. The expression of SCF mRNA and c-Kit protein in intestinal tissue was investigated by RT-PCR and immunohistochemistry. Compared with the sham-operation group, the relative expression of SCF mRNA and the expression of c-Kit protein in intestinal tissue were significantly decreased in both obstruction groups. Levels decreased gradually with the prolongation of obstruction time, and significantly decreased on the 3rd day after obstruction (P<0.05). Immunohistochemical staining of the small intestine showed that the number of ICC in the sham-operation group was the highest, and they were gradually decreased with the extension of obstruction time in the M1 to M3 groups. There was a significant difference between groups (P<0.05). Intestinal obstruction caused a decrease in the concentrations of SCF mRNA and c-Kit protein in ICC. With the prolongation of intestinal obstruction, the number of ICCs gradually decreased.

Asunto(s)

Células Intersticiales de Cajal/metabolismo , Obstrucción Intestinal/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Mensajero/metabolismo , Factor de Células Madre/metabolismo , Animales , Modelos Animales de Enfermedad , Inmunohistoquímica , Células Intersticiales de Cajal/patología , Obstrucción Intestinal/patología , Masculino , Fenotipo , Ratas , Ratas Wistar

RESUMEN

The objective was to study the effect of mechanical intestinal obstruction in rats on the phenotype of interstitial cells of Cajal (ICC). Healthy Wistar rats were randomly divided into sham-operation group (C), one day obstruction group (M1), two days obstruction group (M2), and three days obstruction group (M3), with 10 rats in each group. The expression of SCF mRNA and c-Kit protein in intestinal tissue was investigated by RT-PCR and immunohistochemistry. Compared with the sham-operation group, the relative expression of SCF mRNA and the expression of c-Kit protein in intestinal tissue were significantly decreased in both obstruction groups. Levels decreased gradually with the prolongation of obstruction time, and significantly decreased on the 3rd day after obstruction (P<0.05). Immunohistochemical staining of the small intestine showed that the number of ICC in the sham-operation group was the highest, and they were gradually decreased with the extension of obstruction time in the M1 to M3 groups. There was a significant difference between groups (P<0.05). Intestinal obstruction caused a decrease in the concentrations of SCF mRNA and c-Kit protein in ICC. With the prolongation of intestinal obstruction, the number of ICCs gradually decreased.

Asunto(s)

Animales , Masculino , Ratas , ARN Mensajero/metabolismo , Factor de Células Madre/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Intersticiales de Cajal/metabolismo , Obstrucción Intestinal/metabolismo , Fenotipo , Inmunohistoquímica , Ratas Wistar , Modelos Animales de Enfermedad , Células Intersticiales de Cajal/patología , Obstrucción Intestinal/patología

RESUMEN

Ulcerative colitis is a chronic inflammatory disease of the colon where intestinal motility is disturbed. Interstitial cells of Cajal (ICC) are required to maintain normal intestinal motility. In the present study, we assessed the effect of tumor necrosis factor-alpha (TNF-α) on viability and apoptosis of ICC, as well as on the expression of stem cell factor (SCF), ghrelin, and substance P. ICC were derived from the small intestines of Swiss albino mice. Cell viability and apoptosis were measured using CCK-8 assay and flow cytometry, respectively. ELISA was used to measure the concentrations of IL-1ß, IL-6, ghrelin, substance P, and endothelin-1. Quantitative RT-PCR was used to measure the expression of SCF. Western blotting was used to measure the expression of apoptosis-related proteins, interleukins, SCF, and NF-κB signaling pathway proteins. TNF-α induced inflammatory injury in ICC by decreasing cell viability and increasing apoptosis and levels of IL-1ß and IL-6. TNF-α decreased the levels of SCF, ghrelin, and substance P, but had no effect on endothelin-1. TNF-α down-regulated expressions of SCF, ghrelin, and substance P by activating the NF-κB pathway in ICC. In conclusion, TNF-α down-regulated the expressions of SCF, ghrelin, and substance P via the activation of the NF-κB pathway in ICC.

Asunto(s)

Ghrelina/metabolismo , Células Intersticiales de Cajal/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Células Madre/metabolismo , Sustancia P/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Motilidad Gastrointestinal/efectos de los fármacos , Ghrelina/antagonistas & inhibidores , Células Intersticiales de Cajal/metabolismo , Masculino , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos

RESUMEN

Ulcerative colitis is a chronic inflammatory disease of the colon where intestinal motility is disturbed. Interstitial cells of Cajal (ICC) are required to maintain normal intestinal motility. In the present study, we assessed the effect of tumor necrosis factor-alpha (TNF-α) on viability and apoptosis of ICC, as well as on the expression of stem cell factor (SCF), ghrelin, and substance P. ICC were derived from the small intestines of Swiss albino mice. Cell viability and apoptosis were measured using CCK-8 assay and flow cytometry, respectively. ELISA was used to measure the concentrations of IL-1β, IL-6, ghrelin, substance P, and endothelin-1. Quantitative RT-PCR was used to measure the expression of SCF. Western blotting was used to measure the expression of apoptosis-related proteins, interleukins, SCF, and NF-κB signaling pathway proteins. TNF-α induced inflammatory injury in ICC by decreasing cell viability and increasing apoptosis and levels of IL-1β and IL-6. TNF-α decreased the levels of SCF, ghrelin, and substance P, but had no effect on endothelin-1. TNF-α down-regulated expressions of SCF, ghrelin, and substance P by activating the NF-κB pathway in ICC. In conclusion, TNF-α down-regulated the expressions of SCF, ghrelin, and substance P via the activation of the NF-κB pathway in ICC.

Asunto(s)

Animales , Masculino , Ratones , Ghrelina/metabolismo , Células Intersticiales de Cajal/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Células Madre/metabolismo , Sustancia P/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Motilidad Gastrointestinal/efectos de los fármacos , Ghrelina/antagonistas & inhibidores , Células Intersticiales de Cajal/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos

RESUMEN

As lesões periapicais crônicas estão entre as mais frequentes do complexo maxilofacial, porém o perfil inflamatório dessas lesões é pouco compreendido, tanto do ponto de vista da caracterização celular como da expressão de citocinas. Cistos e granulomas periapicais compõem dois terços dessas lesões inflamatórias em região de mandíbula, onde são mais frequentes. O presente estudo propôs-se a estudar e avaliar a expressão imuno-histoquímica de CD1a+ (marcador de células dendríticas imaturas) e de FoxP3+ (marcador de linfócitos T reguladores) e verificar a presença de mastócitos em granulomas periapicais, cistos radiculares e residuais. Foram selecionados 73 casos, sendo 30 de granulomas periapicais, 29 de cistos radiculares e 14 de cistos residuais, dos arquivos do Serviço de Patologia Cirúrgica Oral e Maxilofacial do Departamento de Estomatologia da Faculdade de Odontologia da Universidade de São Paulo. Todos os grupos foram submetidos a análise morfológica, para classificação do infiltrado inflamatório e espessura epitelial, análise imuno-histoquímica, para detecção e contagem de células dendríticas e linfócitos T reguladores e coloração com azul de toluidina para contagem de mastócitos nas lesões periapicais crônicas. A análise morfológica revelou que a presença de infiltrado inflamatório grau I foi mais comum nos cistos periapicais. A gradação II e III foi mais comumente encontrada em cistos radiculares e granulomas periapicais. A avaliação da espessura epitelial mostrou que os epitélios atrófico e hipertrófico se apresentaram majoritariamente em cistos radiculares. Não houve diferenças estatisticamente significantes em relação ao infiltrado inflamatório e espessura epitelial nas lesões periapicais crônicas estudadas (p>0,05). A avaliação da contagem do número de células dendríticas (CD1a+) apresentou um valor médio maior em cistos radiculares (8,16 células/0,2mm2) (p<0,001) e o número médio de linfócitos T reguladores (FoxP3+) também foi maior em cistos radiculares (5,910 células/0,2mm2) (p<0,05). Na avaliação do número de mastócitos, os cistos radiculares apresentaram maior número médio dessas células do que as outras lesões periapicais (12.68 células/0,2mm2) (p<0,001). A avaliação da correlação entre infiltrado inflamatório e imunomarcação mostrou que houve diferença estatisticamente significante na correlação entre infiltrado inflamatório e células CD1a+ em granulomas periapicais (p<0,001). A medida que a gradação do infiltrado inflamatório aumentou, o número células CD1a+ diminuiu. E a correlação entre espessura epitelial e imunomarcação das células mostrou que a presença de epitélio hipertrófico em cistos radiculares apresentou maior densidade de células CD1a+. Não houve correlação estatisticamente significante da presença de linfócitos Treg e a gradação do infiltrado inflamatório nem da espessura epitelial. Todos esses resultados foram estatisticamente significativos (p<0,05). A concentração de células dendríticas imaturas e linfócitos T reguladores desempenham um papel importante no controle do microambiente inflamatório nos granulomas periapicais e cistos radiculares, respectivamente. A presença de mastócitos nos cistos radiculares pode estar associada à progressão, expansão da lesão e reabsorção óssea.

Asunto(s)

Inmunohistoquímica , Factor de Células Madre , Sarcoma de Células Dendríticas Foliculares

RESUMEN

Homeostatic cell turnover has been extensively characterized in mammals. In their adult tissues, lost or aging differentiated cells are replenished by a self-renewing cohort of stem cells. The stem cells have been particularly well studied in the intestine and are clearly identified by the expression of marker genes including Lgr5 and Bmi1. It is, however, unknown if the established principles of tissue renewal learned from mammals would be operating in non-mammalian systems. Here, we study homeostatic cell turnover in the sea cucumber digestive tube, the organ with high tissue plasticity even in adult animals. Both the luminal epithelium and mesothelium express orthologs of mammalian Lgr5 and Bmi1. However, unlike in mammals, there is no segregation of these positively labeled cells to specific regions in the luminal epithelium, where most of the cell proliferation would take place. In the mesothelium, the cells expressing the stem cell markers are tentatively identified as peritoneocytes. There are significant differences among the five anatomical gut regions in cell renewal dynamics and stem factor expression. The cloaca differs from the rest of the digestive tube as the region with the highest expression of the Lgr5 ortholog, lowest level of Bmi1 and the longest retention of BrdU-labeled cells.

Asunto(s)

Células Epiteliales/metabolismo , Tracto Gastrointestinal/metabolismo , Complejo Represivo Polycomb 1/biosíntesis , Receptores Acoplados a Proteínas G/biosíntesis , Pepinos de Mar/metabolismo , Factor de Células Madre/biosíntesis , Células Madre/metabolismo , Animales , Proliferación Celular , Epitelio/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/genética , Complejo Represivo Polycomb 1/genética , Receptores Acoplados a Proteínas G/genética , Células Madre/citología

RESUMEN

Cell migration of normal cells is tightly regulated. However, tumor cells are exposed to a modified microenvironment that promotes cell migration. Invasive migration of tumor cells is stimulated by receptor tyrosine kinases (RTKs) and is regulated by growth factors. Erythropoietin (Epo) is a glycoprotein hormone that regulates erythropoiesis and is also known to be a potent chemotactic agent that induces cell migration by binding to its receptor (EpoR). Expression of EpoR has been documented in tumor cells, and the potential of Epo to induce cell migration has been explored. Stem cell factor (SCF) is a cytokine that synergizes the effects of Epo during erythropoiesis. SCF is the ligand of c-Kit, a member of the RTKs family. Molecular activity of RTKs is a primary stimulus of cell motility. Thus, expression of the SCF/c-Kit axis is associated with cell migration. In this chapter, we summarize data describing the potential effect of Epo/EpoR and SCF/c-Kit as promoters of cancer cell migration. We also integrate recent findings on molecular mechanisms of Epo/EpoR- and SCF/c-Kit-mediated migration described in various cancer models.

Asunto(s)

Movimiento Celular/fisiología , Eritropoyetina/metabolismo , Neoplasias/metabolismo , Factor de Células Madre/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos

RESUMEN

Los avances en la medicina regenerativa han sido evidentes en los últimos años y esto se ha obtenido por los nuevos conocimientos alcanzados en relación con las células madre, cuyo uso en la terapia de reemplazo ha dado lugar a una nueva era en la medicina. A tales efectos se realizó una revisión bibliográfica con el objetivo de difundir sus generalidades y aplicaciones, así como lo relacionado con las investigaciones básicas que se realizan en ese campo y los principales logros obtenidos. La posibilidad de expansión y diferenciación de dichas células, permite obtener un número suficiente de estas, lo cual ayuda al desarrollo de la terapia celular

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Advances in regenerative medicine have been evident in the last years and this has been obtained due to the new knowledge related to stem cells, which use in the replacement therapy has given rise to a new age in medicine. To such effects, a literature review was carried out aimed at spreading its generalities and implementations, as well as everything related to the basic investigations carried out in this field and the main achievements obtained. The possibility of expansion and differentiation of such cells, allow to obtain enough quantitiy of them, which helps the development of cell therapy

Asunto(s)

Humanos , Masculino , Femenino , Células Madre , Medicina Regenerativa , Tratamiento Basado en Trasplante de Células y Tejidos , Factor de Células Madre/uso terapéutico

RESUMEN

Stem cell factor (SCF), an important stem cell cytokine, has multiple functions. Fibroblasts (FBs), mature mast cells, endothelial cells (ECs), and eosinophil granulocytes can produce SCF in the inflammatory process. Therefore, we aimed to observe SCF expression in FBs, ECs, and macrophages (MPs) in periapical tissues in human chronic periapical disease and investigate the effects of cells expressing SCF in pathogenesis of the disease. Healthy (N = 20), periapical cyst (N = 15), and periapical granuloma (N = 15) tissues were fixed in 10% formalin for 48 h, embedded in paraffin, and stained with hematoxylin and eosin to observe histological changes. SCF expression was observed in FBs, ECs, and MPs in periapical tissues by double immunofluorescence. CD334, CD31, and CD14 are specific markers of FBs, ECs, and MPs, respectively. Results showed that densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs were significantly increased in periapical tissue groups (P < 0.01). There were no significant differences in CD334-SCF double-positive FB and CD31-SCF double-positive EC levels between the two periapical tissue groups (P > 0.05). CD14-SCF double-positive MP density was considerably higher in periapical granulomas than in cysts (P < 0.01). FB, EC, and MP levels were significantly high and densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs improved considerably in chronic periapical tissues, suggesting that the cells might be related to occurrence, development, and pathogenesis of chronic periapical disease.

Asunto(s)

Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Macrófagos/metabolismo , Enfermedades Periapicales/metabolismo , Tejido Periapical/metabolismo , Factor de Células Madre/biosíntesis , Adulto , Anciano , Citocinas/metabolismo , Células Endoteliales/patología , Femenino , Fibroblastos/patología , Humanos , Macrófagos/patología , Masculino , Mastocitos/metabolismo , Mastocitos/patología , Persona de Mediana Edad , Enfermedades Periapicales/patología , Granuloma Periapical/metabolismo , Granuloma Periapical/patología , Tejido Periapical/patología , Quiste Radicular/metabolismo , Quiste Radicular/patología , Factor de Células Madre/metabolismo

RESUMEN

In vitro maturation (IVM) of oocytes in cattle is inefficient, and there is great interest in the development of approaches to improve maturation and fertilization rates. Intraovarian signalling molecules are being explored as potential additives to IVM media. One such factor is kit ligand (KITL), which stimulates the growth of oocytes. We determined if KITL enhances oocyte maturation in cattle. The two main isoforms of KITL (KITL1 and KITL2) were expressed in bovine cumulus-oocyte complexes (COC), and levels of mRNA increased during FSH-stimulated IVM. The addition of KITL to the culture medium increased the percentage of oocytes that reached meiosis II but did not affect cumulus expansion after 22 h of IVM. Addition of KITL reduced the levels of mRNA encoding natriuretic peptide precursor C (NPPC), a protein that holds oocytes in meiotic arrest, and increased the levels of mRNA encoding YBX2, an oocyte-specific factor involved in meiosis. Removal of the oocyte from the COC resulted in increased KITL mRNA levels and decreased NPPC mRNA levels in cumulus cells, and addition of denuded oocytes reversed these effects. Taken together, our results suggest that KITL enhances bovine oocyte nuclear maturation through a mechanism that involves NPPC, and that the oocyte regulates cumulus expression of KITL mRNA.

Asunto(s)

Células del Cúmulo/citología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Péptido Natriurético Tipo-C/metabolismo , Oocitos/citología , Oogénesis/fisiología , Factor de Células Madre/metabolismo , Animales , Bovinos , Células del Cúmulo/metabolismo , Femenino , Fertilización In Vitro , Meiosis/fisiología , Oocitos/metabolismo

RESUMEN

The aim of this study was to evaluate the mechanism involved in the stem cell factor (SCF)-induced production of fibroblast growth factor-2 (FGF-2), transforming growth factor-ß1 (TGF-ß1), and chemokine (C-C motif) ligand 3 (CCL3) in tracheal smooth muscle cells (tSMCs) and the signaling pathway involved in the process. tSMC primary cultures were stimulated with SCF and evaluated at 24 h. Cells treated with specific antibodies did not show any immunolabeling for cytokeratin or fibroblast activation protein, but were positive for α-smooth muscle actin, indicating the purity of the primary cell line. Western blot analysis showed constitutive phosphorylation of c-Kit, as well as increased total protein and phosphorylated c-Kit levels in tSMCs after SCF stimulation. Flow cytometry analysis also showed an increase in cell-surface c-Kit expression in the presence of SCF. SCF induced TGF-ß mRNA expression in tSMCs, as well as the production of TGF-ß1, CCL3, and FGF-2. Pretreatment with anti-CCL3 antibody blocked TGF-ß1 expression and partially inhibited FGF-2 production. On the other hand, anti-c-Kit antibody blocked TGF-ß1 expression and FGF-2 production. Thus, TGF-ß1 and FGF-2 production were mediated by CCL3 production through c-Kit. Pretreatment with mitogen-activated protein kinase kinase 1, p38, and Jun N-terminal kinase inhibitors showed that the effects mediated by SCF were involved with the modulation of mitogen-activated protein kinase (MAPK) pathways. Development of inhibitors targeting CCL3 through MAPK activation could thus be an attractive strategy to inhibit tSMC activation during asthma.

Asunto(s)

Quimiocina CCL3/metabolismo , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/metabolismo , Tráquea/citología , Factor de Crecimiento Transformador beta1/biosíntesis , Animales , Quimiocina CCL3/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Expresión Génica , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Células Madre/farmacología , Factor de Crecimiento Transformador beta1/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo